**2.** *Lachancea thermotolerans* **and** *Hanseniaspora* **spp.**

Yeast selection is a powerful tool to search for new strains with improved features that can enhance the sensory profile of wine or facilitate the technological process. Historically, vinifications have been performed with *Saccharomyces cerevisiae*, however, current enology is strongly focused on non-*Saccharomyces yeasts* [5]. Species such as: *Metschnikowia pulcherrima* [6], *Brettanomyces bruxellensis* [7], *Torulaspora delbrueckii* [8], *Aureobasidium pullulans* [9], *Hanseniaspora/ Kloeckera* spp. [10], *Candida stellata* [11], *Saccharomycodes ludwigii* [12], *Starmerella bacillaris* [13], *Schizosaccharomyces pombe* [14], *Zygosaccharomyces rouxii* [15], *Wickerhamomyces anomalus* [16], *Lachancea thermotolerans* [17]. Most of them were used for their positive impact on wine aroma, flavor, mouthfeel, or color, and some of them were studied for their spoilage activity that may negatively affect wine quality.

This chapter is focused on the species *Lachancea thermotolerans* (Lt) (**Figure 1**) and the genus *Hanseniaspora* (H) spp. (**Figure 2**) because of their interesting behavior to improve the sensory profile and enhance the freshness of wines from warm areas. The main feature of Lt is the effective acidification by the formation of lactic acid from sugars [17]. Several lactate dehydrogenase sequences have been observed in the genome of Lt. Its morphology is similar to that of *Saccharomyces cerevisiae* (Sc) with ellipsoidal geometry and multipolar budding (**Figure 1**), although Lt shows a slightly smaller size. The use of Lt for wine acidification, pH control, and freshness improvement has been described

#### **Figure 1.**

*Optical microscopy of* Lachancea thermotolerans *(left) compared with* Saccharomyces cerevisiae *(right) both at different growth stages. Both species show an ellipsoidal shape with multipolar budding.*

*pH Control and Aroma Improvement Using the Non-*Saccharomyces Lachancea*… DOI: http://dx.doi.org/10.5772/intechopen.100538*

**Figure 2.**

*Optical microscopy of* Hanseniaspora vineae*, apiculate yeast with polar budding. Cells are in different stages of growth.*

in several works [18–24]. Acidification and pH control in warm areas is critical for wine quality and stability. A low pH not only produces fresher wines with a better sensory profile and improved consumer perception but also increases wine stability at the chemical and microbiological levels. So, wines with low pH are safer and more stable, and, as mentioned before, pH also favors higher molecular SO2 content with higher antimicrobial and antioxidant performance. Therefore, biological acidification is a way to protect the wine and allows the reduction of SO2 levels. The effect on molecular SO2 at low pH has an impact on reducing the levels of spoilage microorganisms and, as a consequence, lowering the production of off-flavors and toxic molecules such as biogenic amines and others, thus producing safer and cleaner wines [25].

Lt shows a medium fermentative power with some strains reaching 9–10% vol. in ethanol [17]. In addition, Lt has shown other interesting features such as moderate volatile acidity [18, 22], even when used simultaneously with other species (*Metschnikowia pulcherrima*, *Hanseniaspora vineae*, *Torulaspora delbrueckii*) [23], and also reduction of volatile acidity levels in some conditions [26]. Furthermore, the positive role in the formation of thiol compounds in Sauvignon blanc has been described, releasing higher values of 3-Mercapto-1-hexanol (3MH) than the control yeast *Saccharomyces cerevisiae* (Sc) and significant contents of 4-Mercapto-4-methyl-2-pentanone (4MMP) compared to other non-*Saccharomyces* although, in this case, lower than Sc [27]. These thiol compounds are responsible for box tree (4MMP) and tropical fruit aroma (3MH) in wines that increase their complexity [28, 29]. Lt is a low producer of medium-chain fatty acids and their esters, therefore avoid heavy smells and flatness, which helps improve freshness [24].

The low pH produced by the intense biological acidification of Lt also has a positive effect on the color of white wine showing a bright and clean appearance and delaying the browning processes. This effect on browning is also evidenced by the higher levels of molecular SO2 obtained at low pH which produces an intense antioxidant effect. Concerning red wine color, this reduction in pH favors an increase in color intensity by hyperchromic effect, but it also favors the stability of anthocyanins [30, 31].

In addition, we have observed that some Lt strains have an impact on wine structure, producing softer and full-bodied wines. However, this is not a typical feature of the Lt species, but only of some specific strains. It can be interesting to select these strains to achieve a good balance between acidity and mouthfeel.

*Hanseniaspora* species (*vineae*, *opuntiae*, *uvarum*, *guilliermondii*, *osmophila*, *valbyensis,* and others) are lemon-shaped apiculate yeasts with polar budding (**Figure 2**) that are typically found in grape juices at the onset of alcoholic fermentation [10], being included in the predominant indigenous yeast population of grapes. Most of them have a low fermentative power around or below 4% vol. However, some of them such as *H. vineae* can reach around 10% vol. [10].

Normally, *Hanseniaspora* spp. have been described as high producers of volatile acidity and have been removed from wine fermentation using SO2 because of their high sensitivity to this antimicrobial agent. However, acetic acid production is quite variable among strains and some of them can reach values similar to those of Sc [32]. Some species such as *H. vineae* or *H. opuntiae* also show low values (<0.4 g/L) that can be comparable or lower than Sc [33, 34].

Several enzymatic activities have been described in *Hanseniaspora* spp., being especially interesting concerning aroma the expression of the β-D-glucosidase activity to release the free terpenes from their conjugated glucosides [35]. The latter compounds are found in higher concentrations in terpene-rich varieties, but due to their low volatility, they are odorless compounds. The use of non-*Saccharomyces* species with β-D-glucosidase activity is a way to increase wine aroma by releasing free terpenols.

*Hanseniaspora vineae* (Hv, anamorph sp. *Kloeckera africana*) [36] is one of the most interesting and trending species in enology, due to its medium-high fermentative power (up to 10% vol), its low volatile acidity, but especially for its high impact on wine aroma and structure. Some extra nutritional requirements have been described especially in thiamine, pantothenic acid, and YAN (yeast assimilable nitrogen) supplementation to avoid stuck or sluggish fermentations [10, 37]. The molecular proximity of Hv to Sc in phylogenetic trees is higher than that of other *Hanseniaspora* spp. (*H. opuntiae*, *H. guilliermondii*, *H., uvarum*) (**Figure 3**).

In addition to its interesting fermentative behavior with good implantation and suitable fermentation yield, Hv is useful to modulate the sensory profile of wines. The impact on the aroma is quite significant due to the formation of benzenoid compounds *de novo* by the chorismate-prephenate metabolic pathway (**Figure 4**). This pathway uses sugars as precursors and leads to the formation of floral benzenoid acetic esters such as benzyl acetate and 2-phenylethyl acetate [10, 36, 38, 39]. The production of 2-phenylethyl acetate among other fermentative compounds can

#### **Figure 3.**

*Phylogenetic relationships among wine yeast species based on analysis of D1/D2 LSU rRNA gene sequences. The evolutionary history was inferred using the maximum likelihood method based on the Tamura-Nei model in MEGA7. GenBank access numbers follow strain numbers:* Saccharomyces cerevisiae *NRRL Y12632/ AY048154;* Lachancea thermotolerans *CBS 2803/KY108273;* Hanseniaspora uvarum *NRRL Y-1614/U84229;*  Hanseniaspora opuntiae *CBS 8733/AJ512453;* Hanseniaspora vineae *NRRL Y-17529/U84224;* Hanseniaspora guilliermondii *NRRL Y1625/U84230.*

*pH Control and Aroma Improvement Using the Non-*Saccharomyces Lachancea*… DOI: http://dx.doi.org/10.5772/intechopen.100538*

**Figure 4.**

*De novo formation of floral esters by* Hanseniaspora *spp. from sugars via the chorismate-prephenatemandelate pathway. 2-phenylethyl acetate with rose petal aroma descriptor and benzyl acetate with jasmine aroma descriptor.*

separate, by PCA statistical analysis, the aromatic profile of Hv from Sc [34]. Benzyl alcohol concentrations in the fermentation of 11 Hv strains can reach x20-x200 the typical concentrations produced by Sc [38]. Benzyl acetate is the impact aroma of jasmine flowers and produces floral scents that help improve the sensory profile of wines produced from neutral grape varieties. Another impact compound in terms of floral aroma is 2-phenylethyl acetate, also produced by Hv. Its descriptor is rose petals and produces fresh floral perception in wines increasing complexity. This compound is also produced by other *Hanseniaspora* spp. such as *H. guilliermondii* [40], *H. uvarum* [41], *H. opuntiae* [42].

The impact of Hv on wine aroma is also related to the release or *de novo* formation of terpenes. Terpenes are aromatic compounds with a fruity and floral profile that enhance the aroma complexity and freshness of wines. Some grape varieties (Muscat, Gewürztraminer, Albariño) have terpenes produced by the plant in the form of terpenes bonded to sugars as a way to better translocate the hydrophobic free terpenes through the plant tissues. Bonded terpenes are more polar but less volatile, so less aromatic. Hv can express extracellular β-Dglucosidase releasing free terpenes during fermentation and thus improving the varietal aroma of wines [10, 35, 43]. The β-xylosidase activity has also been described in Hv [43].

*De novo* formation of terpenes from sugars has also been observed in fermentations with Hv. In the fermentation of the neutral variety Macabeo, the formation of a significant concentration of α-terpineol (>100 μg/L) has been observed, but below its sensory threshold [36]. Sequential fermentations with Hv followed by Sc in Albillo grapes have shown much higher concentrations of terpenes (316 μg/L) than with Sc controls (114 μg/L) [44]. Linalool, β-citronellol, and geraniol showed higher concentrations than in the Sc control (>x3, >x4, and > x2 respectively), but also above their respective sensory thresholds [44]. The balsamic terpenes terpinene-4-ol and α-terpineol were also at significantly higher concentrations but below the sensory threshold. Furthermore, several polyoxygenated terpenes showed significantly higher concentrations, but they usually have higher sensory thresholds and, therefore, less impact on the aroma.

Another interesting impact of some *Hanseniaspora* species is the effect on wine structure. Usually, wines fermented by these yeasts show a full-bodied structure and better palatability in the mouth. Fermentation of Macabeo grape must with Hv has shown a sensory profile where tasters perceived improved structure and volume [10]. When the contents of cell wall polysaccharides released by Hv were measured by size exclusion chromatography no significant differences were found with Sc. However, the absorbance at 280 nm, which can be correlated with protein, shows higher values especially at the end of fermentation with Hv [34]. When aging on lees (AOL) is extended for several months, there are no differences between Hv and Sc control. The use of size exclusion chromatography showed slightly higher molecular sizes in the polysaccharides released by Hv that may influence the more intense mouthfeel [44].
